Component of bromelain

ABSTRACT

The invention relates to a component of bromelain which is largely responsible for the ability of bromelain to interrupt the MAP kinase cascade. The component contains ananain and comosain and is useful in the treatment or prevention of diseases and conditions mediated by T cell activation or by activation of the MAP kinase pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/750,210, filed Dec. 29, 2000 (now abandoned), which was publishedAug. 1, 2002 as U.S. Application Publication No. US-2002-0102253-A1,which is a divisional of U.S. application Ser. No. 09/382,688, filedAug. 25, 1999 (now abandoned), which is a continuation of U.S.application Ser. No. 09/380,095, filed Aug. 25, 1999 (now abandoned),which is the U.S. national phase application of InternationalApplication No. PCT/GB98/00590, filed Feb. 25, 1998, which was publishedunder PCT Article 21(2) in English as WIPO Publication No. WO 98/38291on Sep. 3, 1998; which Applications claim priority under 35 U.S.C. §119to the following United Kingdom Patent Application Nos. GB 9703850.9,filed Feb. 25, 1997, GB 9703827.7, filed Feb. 25, 1997, GB 9704252.7,filed Feb. 28, 1997, and GB 9706119.6, filed Mar. 25, 1997.

The present invention relates to a component of bromelain. Inparticular, the invention relates to the use of this bromelain componentin medicine, particularly as an anticancer agent and animmunosuppressive agent.

Stem bromelain (bromelain) is the collective name for the proteolyticenzymes found in the tissues of the plant Bromeliaceae. It is a mixtureof various moieties derived from the stem of the pineapple plant (Ananascomosus). Bromelain is known to contain at least five proteolyticenzymes but also non-proteolytic enzymes, including an acid phosphataseand a peroxidase; it may also contain amylase and cellulase activity. Inaddition, various other components are present.

Bromelain has previously been used in the treatment of a variety ofconditions including inflammation and, in particular, it has been usedin the treatment of diarrhoea. The use of bromelain in the treatment ofinfectious diarrhoea is described in WO-A-9301800, where it is suggestedthat bromelain works by destroying intestinal receptors for pathogens byproteolysis, and in WO-A-8801506, which teaches that bromelain detachespathogens from intestinal receptors.

Taussig et al, Planta Medica, 1985, 538-539 and Maurer et al, PlantaMedica, 1988, 377-381 both suggest that bromelain may be of use ininhibiting tumour growth. U.S. Pat. No. 5,223,406, DE-A-4302060 andJP-A-59225122 also teach the use of bromelain in the treatment ofcancer. U.S. Pat. No. 5,223,406 teaches that bromelain is capable ofinducing tumour necrosis factor (TNF) while DE-A4302060 teaches thatbromelain can prevent metastasis by the structural modification of thetumour surface protein CD44.

In WO-A-9400147, various experiments were described which demonstratethat proteolytic enzymes and, in particular, bromelain, are capable ofinhibiting secretion. The application also discloses that bromelain canreduce toxin binding activity and can inhibit the secretory effect oftoxins such as heat labile toxin (LT) and cholera toxin (CT) and alsotoxins such as heat stable toxin (ST). This is in spite of the fact thatST has a very different mode of action from LT and CT. Theseobservations were explained by the fact that one component of thebromelain mixture, stem bromelain protease, appears to be capable ofmodulating cyclic nucleotide pathways and this is discussed further inWO-A-9500169. In addition, bromelain has also been demonstrated toinhibit secretion caused by the calcium dependent pathway.

The present inventors have studied the varied biological effects ofbromelain and, in particular, its effects in a well documented model ofintracellular signal transduction, namely T cell receptor (TCR)/CD3signalling and IL-2 production. Significant progress over recent yearshas led to the understanding of biochemical events which occur followingTCR engagement (reviewed Cantrell, Annu. Rev. Immunol. 14, 259-274,(1996)), therefore TCR signalling provides an excellent model forelucidation of the effects of biologically active compounds. Effective Tcell activation requires two signals. The first signal is generated bythe TCR/CD3 complex after engagement with antigen peptide presented bythe major histocompatibility complex (MHC) expressed on antigenpresenting cells (APC) (Cantrell, 1996). The second, costimulatorysignal is generated by ligation of CD28 receptors on T cells with the B7family of ligands on APC. A key element in the signalling pathwayinvolved in transducing receptor-initiated signals to the nucleus is thefamily of mitogen-activated protein kinases (MAPk). The best studied ofthese kinases are the extracellular signal-regulated protein kinases(ERK)-1 and ERK-2 (also referred to as p44^(MAPk) and p42^(MAPk),respectively). ERK's are serine/threonine kinases that are activatedwhen phosphorylated on tyrosine and threonine residues. In vitro, thisactivation is reversed if either residue is dephosphorylated. Arelatively newly discovered member of the MAPk family are c-JunNH₂-terminal kinases (JNKs) which exist as 46 kDa and 55 kDa forms thatalso require phosphorylation for activation. ERK activation is dependenton p56^(Lck) and coupling of the TCR/CD3 complex to p21^(Ras), withsubsequent activation of the Raf-1/MEK1/ERK kinase cascade. JNKactivation also requires p21%, as well as signals generated by the CD28costimulatory receptor which activate GTP (guanosinetriphosphate)-binding proteins (such as Rac1 or Cdc42) that induce thePAK/MEKK/SEK/JNK kinase cascade. Activated ERK phosphorylates Elk-1,which in turn, mediates induction of c-fos activity followingphosphorylation of c-jun by JNK. Activated c-fos and c-jun combine toform the AP-1 protein required for IL-2 synthesis. The above events aresummarised in FIG. 1. All the above-mentioned signalling events requiretyrosine phosphorylation, as inhibitors of protein tyrosine kinases(PTKs) inhibit many events associated with TCR stimulation, including Tcell activation and IL-2 production.

In WO-A-9600082, we showed that bromelain could inhibit tyrosinephosphorylation and activation of ERK-2 in T cells stimulated via theTCR, or with combined phorbol ester plus calcium ionophore. We have nowfound that, in association with decreased ERK activity, bromelaindecreased IL-2, IL-4 and IFN-γ mRNA accumulation in T cells stimulatedwith phorbol ester and ionophore, but did not affect cytokine mRNAaccumulation in cells stimulated via the TCR. This data suggests theexistence of a TCR-activated, ERK-independent pathway involved incytokine production in T cells.

From the prior art, it is clear that bromelain is a mixture which has avariety of different physiological effects. Not all of the components ofthe bromelain mixture have been characterised and so, except for stembromelain protease, whose activity we have described, it is not clearwhich of the components is responsible for which of the variousdifferent effects of bromelain. This is, of course, a major disadvantageif the bromelain mixture is to be administered as a pharmaceuticalbecause while one component of bromelain might give the desired effect,there may well be unwanted side effects arising from the action of someother component of the bromelain mixture.

It would therefore be beneficial if individual components of bromelaingiving rise to particular medicinal activities could be isolated andadministered separately so as to lessen the possibility of side effects.We have now identified an active fraction of crude bromelain which isresponsible for its ability to inhibit ERK activation, and thereforeblock the MAP kinase pathway. Although not a single protein, thisfraction consists of only a few components and so the possibility ofside effects when it is administered to patients is greatly reducedcompared with crude bromelain.

The fraction of the invention, which the inventors have designated CCS,may be isolated from the bromelain mixture by conventional methods, forexample by chromatography. High performance liquid chromatograpy (HPLC)is suitable for the purpose and particularly good separation ofbromelain proteins may be achieved by fast protein liquid chromatography(FPLC™) using a column packing material such as S-sepharose. As will bedescribed in more detail in the examples, in chromatography onS-sepharose using a linear gradient of 0 to 0.8M sodium chloride inacetate buffer over 300 ml, the protein of the present invention was thelast double peak off the column.

In a first aspect of the present invention, there is provided acomponent of bromelain which contains proteins having molecular weightsof about 15.07 kDa, 25.85 kDa and 27.45 kDa as determined by SDS-PAGE,has isoelectric points of 10.4 and 10.45 and is obtainable by thefollowing method:

-   -   i. dissolving bromelain in acetate buffer at pH 5.0;    -   ii. separating the components of the bromelain by fast flow high        performance liquid chromatography on S-sepharose eluting with a        linear gradient of 0 to 0.8 M sodium chloride in acetate buffer        over 300 ml;    -   iii. collecting the fraction corresponding to the final double        peak off the column; and    -   iv. isolating the protein from the fraction collected in (iii).

This fraction, termed CCS, has been found to have a number ofpotentially useful activities. Firstly, we have found that it blocksERK-2 phosphorylation, and therefore the MAP kinase cascade. Inaddition, it blocked IL-2 production and CD4⁺ T cell proliferation.However, CCS did not affect splenocyte proliferation, which suggeststhat it has a selective mode of action. CCS also differentially blockedgrowth of human tumour cell lines including ovarian, lung, colon,melanoma and breast tumours. The differential activity of CCS againstthe different cell lines further suggests that CCS has a selective modeof action and that it may also act as an anti-cancer agent. Theinhibitory effect of CCS on ERK-2 is dependent on its proteolyticactivity, since E-64, a selective cysteine protease inhibitor, couldabrogate the effect of CCS.

Although in WO-A-9724138, we stated that proteases in general arecapable of decreasing MAP kinase activation, we have now found that thisis not the case as trypsin does not abrogate T cell signalling and,indeed, in other studies, has been shown to increase MAPk activation(Belham et al, 1996, Biochem. J., 320, 939-946). Thrombin, a proteaseinvolved in the blood coagulation cascade, has also been shown toincrease MAP kinase activation (Vouret-Craviari et al, 1993, Biochem.J., 289, 209-214). The inventors have now also shown that otherproteases contained within the crude bromelain mixture do not block theactivation of the MAP kinase pathway.

It is possible that the effects of CCS on the MAP kinase pathway in Tcells are mediated by specific proteolytic effects at the cell surface.It is known that bromelain cleaves the CD45 RA isoform and selectivelyremoves other surface molecules from human PBMCs. Bromelain alsopartially removes CD4 from T cell surfaces. Since CD45 and CD4 play anobligate stimulatory role in TCR-mediated T cell activation, CCS mayinterfere with TCR signalling by affecting these molecules. Although theimportance of CD45 and CD4 is well recognised for TCR-initiated signaltransduction, it is possible to bypass their requirements for T cellactivation by the use of phorbol ester and calcium ionophore. Use ofcombined phorbol esters and ionophore restores normal function to Tcells which have been made refractory to TCR stimulation by the use oftyrosine kinase inhibitors or which are CD45 or p56^(Lck) deficient.

However, in the present study, the inventors have shown that normalfunction is not restored to T cells pre-treated with CCS when they aretreated with PMA plus ionophore. The inhibitory effect of CCS on ERK-2is thus not thought to be mediated via effects on CD45 or CD4 on Tcells. CCS possibly affects an as yet unidentified surface molecule,which, in turn, affects the MAP kinase pathway. The inhibitory effect ofCCS on cytokine production is thus not thought to be mediated via itseffects on CD45 or CD4 on T cells. The inhibitory effect of CCS on Tcell signal transduction was not because of toxicity of the compound,since CCS did not affect splenocyte or GA15 cell viability. Theviability of the cells was not significantly affected by culture in thepresence of CCS for periods of time greater than 48 hours.

Since we have shown that fraction CCS from crude bromelain blocksactivation of the MAP kinase pathway and blocks T cell activation, CCSmay be of use in the treatment of T cell-mediated diseases.

In addition to its importance for IL-2 production and T cell activation,the MAP kinase pathway is also important for the production of growthfactors such as epidermal growth factor (EGF), platelet derived growthfactor (PGDF) and insulin-like growth factor (IGF). CCS will thereforeblock the production of these, and other, growth factors and theproduction of other cytokines such as IL4, IFN-γ, GM-GSF and many more.

Also, as briefly mentioned above, CCS is likely to be of use in thetreatment of cancer.

Thus, CCS may be of use in a method for the treatment or prevention ofdiseases or conditions mediated by:

-   i. activation of T cells;-   ii. activation of the MAP kinase pathway; or-   iii. the production of growth factors or cytokines;-   or in the treatment or prevention of cancer.

In a second aspect of the invention, therefore, there is provided acomponent of bromelain which contains proteins having molecular weightsof about 15.07 kDa, 25.85 kDa and 27.45 kDa as determined by SDS-PAGE,has isoelectric points of 10.4 and 10.45 and is obtainable by thefollowing method:

-   -   i. dissolving bromelain in acetate buffer at pH 5.0;    -   ii. separating the components of the bromelain by fast flow high        performance liquid chromatography on S-sepharose eluting with a        linear gradient of 0 to 0.8 M sodium chloride in acetate buffer        over 300 ml;    -   iii. collecting the fraction corresponding to the final double        peak off the column; and    -   iv. isolating the protein from the fraction collected in (iii)        for use in medicine, particularly in the treatment or prevention        of diseases and conditions mediated by:

-   i. activation of T cells;

-   ii. activation of the MAP kinase pathway; or

-   iii. the production of growth factors or cytokines;

-   or in the treatment or prevention of cancer.

On further analysis of the CCS fraction of bromelain, the presentinventors have found that it comprises more than one component.Sequencing of the proteins in the fraction showed that it consists ofthe cysteine proteases ananain and comosain together with various othercomponents.

Thus, it appears that both ananain and comosain or a mixture of the twomay be responsible for the activity of the CCS fraction of bromelain.

In a further aspect of the invention, therefore, there is provided theuse of ananain, comosain, a mixture of ananain and comosain or the CCSfraction of bromelain in the preparation of an agent for treatment orprevention of diseases or conditions mediated by:

-   i. activation of T cells;-   ii. activation of the MAP kinase pathway; or-   iii. the production of growth factors or cytokines;-   or in the treatment or prevention of cancer.

In our earlier application WO-A-9600082 we discussed the inhibition ofthe MAP kinase cascade by crude bromelain. However, at that time, wewere not able to determine which component of the crude bromelainmixture was responsible for this activity although we speculated that itmight be stem bromelain protease. We have now discovered that inaddition to blocking cyclic nucleotide pathways, stem bromelain proteasedoes have some activity against the MAP kinase pathway. However, it isfar less effective in blocking the MAP kinase cascade than the CCSfraction of bromelain of the present invention. Indeed, we have nowfound that the CCS fraction of bromelain is in the region of ten ordersof magnitude more active than stem bromelain protease in blocking MAPkinase activation.

The activation of the MAP kinase pathway in T cells to produce IL-2 anddrive T cell clonal expansion is an essential component of the immuneresponse. The absence of this process can have fatal consequences, ascan be observed in people with AIDS or genetic mutations which result inT cell defects. However, the activation of T cells can also lead todetrimental consequences. For example, if autoreactive T cells areactivated, autoimmune diseases can result. CCS is therefore likely to beof use in the treatment of autoimmune diseases such as rheumatoidarthritis, type-1 diabetes mellitus, multiple sclerosis, Crohn's diseaseand lupus.

Also, the activation of T cells specific for engrafted tissue can leadto graft or transplant rejection and so CCS may also be of use inpreventing this.

The activation of allergen-specific T cells can cause allergicreactions. Inflammatory cytokines and other cellular products, such ashistamine, are released from cells following exposure to allergens. Therelease of histamine and inflammatory cytokines involves the MAP kinasepathway and so blocking of the MAP kinase pathway with CCS is likely tobe an effective treatment for allergies.

In addition, CCS is likely to be of use in the prevention of toxic shockand other diseases mediated by over production of bacterial endotoxins.Toxic shock is mediated by the production of lipopolysaccharides (LPS)from gram-negative bacteria. LPS triggers the production of TNF-α andinterleukin-1 via activation of the MAP kinase pathway in macrophages.The secretion of these cytokines elicits a cascade of cytokineproduction from other cells of the immune system (including T cells),which leads to leucocytosis, shock, intravascular coagulation and death.

A further use for CCS is in the prevention of programmed cell death(apoptosis). This is a special event whereby cells are stimulated todestroy their own DNA and die. It is an essential event in most immuneresponses (to prevent the accumulation of too many cells), but can alsohave immunosuppressive consequences in some instances, such as in HIVinfection and ageing so that too many cells die and there areinsufficient left to combat infection (Perandones et al, 1993, J.Immunol., 151, 3521-3529). Because the initiation of apoptosis isdependent on specific cell signalling events, including activation ofthe MAP kinase pathway, CCS is likely to be effective in blockingapoptosis.

The continual activation of T cells during chronic disease can also leadto pathological consequences, as can be found in certain chronicparasitic infections, such as chronic granulatomas diseases such astuberculoid leprosy, schistosomiasis and visceral leishmaniasis.Furthermore, the invasion of parasites and pathogens, and theirsubsequent survival in cells, is dependent on these organisms utilisinghost cell signalling pathways (Bliska et al, 1993, Cell, 73, 903-920).For example, Salmonella has been demonstrated to phosphorylate MAPkinase, which allows for the bacteria to become endocytosed bymacrophages (Galan et al, 1992, Nature, 357, 588-589). The bacteria thenproliferate and destroy the cell. Because CCS has been shown to modifyhost signalling pathways, and, in particular, to inhibit MAP kinase,another of its potential applications could be to inhibit invasion byparasites and pathogens and their survival in cells.

CCS may also be of use for the treatment of cancer and, indeed, we haveshown that CCS can block human tumour growth in vitro. The anti-tumourmechanism of action of CCS remains to be determined but seems likely tobe a result of the blocking of activation of the ERK-2 pathway.

As mentioned earlier, MAP kinase activation is dependent on p21^(Ras)and Raf-1, which are important oncogenes. p21^(Ras) and Raf-1 proteinshelp to relay signals from growth factor receptors on the surface ofcells to MAP kinases to stimulate cell proliferation or differentiation.Oncogenic (or mutant) p21^(Ras) or Raf-1 genes produce defectiveproteins that have acquired independence from externally supplied growthfactors and, at the same time, may no longer respond to externalgrowth-inhibitory signals. Mutant p21^(Ras) or Raf-1 proteins are thuspersistently hyperactive and their unbridled catalytic activity has adetrimental effect on the control of cell growth. Oncogenic p21^(Ras) orRaf-1 genes therefore promote cancer and tumour formation by disruptingthe normal controls on cell proliferation and differentiation.Approximately 30% of human cancers have mutations in a p21^(Ras) gene.

Given that signals transmitted by p21^(Ras) and Raf-1 can be blocked viaMAP kinase, CCS would be expected to block cancer and tumour growth. Theprotein fraction of the present invention would therefore be useful fortreating many different types of cancer including solid cancers such asovarian, colon, breast or lung cancer and melanoma as well as non-solidtumours and leukaemia.

The bromelain fraction of the invention will usually be formulatedbefore administration to patients and so, in a further aspect of theinvention there is provided a pharmaceutical or veterinary compositioncomprising the CCS fraction of bromelain together with apharmaceutically or veterinarily acceptable excipient.

The CCS fraction may be administered by a variety of routes includingenteral, for example oral, nasal, buccal, topical or anal administrationor parenteral administration, for example by the intravenous,subcutaneous, intramuscular or intraperitoneal routes.

In many cases, the oral route may be preferred as this is often theroute which patients find most acceptable. The oral route may beparticularly useful if many doses of the protein are required.

When oral administration is chosen, it may be desirable to formulate theCCS fraction in an enteric-coated preparation in order to assist itssurvival through the stomach. Alternatively, another orallyadministrable dosage form may be used, for example a syrup, elixir or ahard or soft gelatin capsule, either of which may be enteric coated.

However, under certain circumstances, it may more convenient to use aparenteral route. For parenteral administration, the protein may beformulated in distilled water or another pharmaceutically acceptablesolvent or suspending agent.

A suitable dose of the CCS fraction to be administered to a patient maybe determined by the clinician. However, as a guide, a suitable dose maybe from about 0.5 to 20 mg per kg of body weight. It is expected that inmost cases, the dose will be from about 1 to 15 mg per kg of body weightand preferably from 1 to 10 mg per kg of body weight. For a man having aweight of about 70 kg, a typical dose would therefore be from about 70to 700 mg.

The invention will now be further described with reference to thefollowing examples and to the drawings in which:

FIG. 1 is a diagrammatic representation of signal transduction eventsassociated with T cell activation that lead to IL-2 production.

FIG. 2 is an ultra violet elution profile of crude bromelain aftercation exchange chromatography on SP Sepharose high performance media.

FIG. 3 is a plot showing the proteolytic activity and the proteincontent of crude bromelain fractions after cation exchangechromatography on SP Sepharose high performance media.

FIG. 4 is an SDS-PAGE of SP Sepharose high performance chromatographypooled fractions run on 4-20% T gradient gels with lanes 1 to 4 and 6 to9 containing proteins CCT, CCV, CCX and CCZ and CCY, CCW, CCU and CCSrespectively and lanes 5 and 10 containing molecular weight markers.

FIG. 5 shows isoelectric focussing of pooled fractions run on pH 3-11gradient gels with Lanes 1, 11 and 12 showing high IEF markers, Lanes 2and 13 showing crude bromelain and Lanes 3 to 10 showing proteins CCT,CCV, CCX, CCZ, CCY, CCW, CCU and CCS respectively.

FIG. 6 is a Western blot using anti-phosphotyrosine mAb whichdemonstrates that CCS reduces tyrosine phosphorylation of p42 kDa(ERK-2) protein. Th0 cells were treated with bromelain fractions (50μg/ml) for 30 min, washed and then stimulated with combined PMA (20ng/ml) and ionophore (1 μM) for 5 min. Unstimulated cells served ascontrols. Cells were then lysed and postnuclear supernatants weresubjected to SDS-PAGE and Western blotting. In this figure, closedsymbols indicate proteins phosphorylated by combined PMA plus ionophore.Open symbols indicate ERK-2 protein reduced by CCS treatment.

FIG. 7 is a Western blot using anti-phosphotyrosine mab whichdemonstrates that CCS increases tyrosine phosphorylation of proteinsubstrates. Th0 cells were treated with CCS, crude bromelain (Brom),stem bromelain protease (SBP) or CCT fraction (50 μg/ml) for 30 min,washed and then stimulated with combined PMA (20 ng/ml) and ionophore (1μM) for 5 min. Unstimulated cells served as controls (Cont). Cells werethen lysed and postnuclear supernatants were subjected to SDS-PAGE andWestern blotting. In this figure, closed symbols indicate proteinsphosphorylated by CCS but not by other treatments. Open symbols indicatephosphoproteins protein reduced by CCS and crude bromelain treatment.

FIG. 8 is a Western blot using anti-phosphotyrosine mAb which shows thatthe inhibitory effect of CCS on ERK-2 is dependent on its proteolyticactivity and occurs in a dose-dependent manner. Th0 cells were treatedwith CCS (0 to 25 μg/ml) or CCS incubated with the selected proteaseinhibitor, E-64, for 30 min. Cells were then washed and then stimulatedwith combined PMA (20 ng/ml) and ionophore (1 μM) for 5 min. Cells werethen lysed and postnuclear supernatants were subjected to SDS-PAGE andWestern blotting. In this figure, closed symbols indicate proteinsphosphorylated by CCS. Open symbols indicate ERK-2 phosphoproteininhibited by active CCS but not by inactivated CCS.

FIG. 9 is an immunoblot which confirms that the 42 kDa phosphoproteininhibited by CCS is ERK-2. Th0 cells were treated with CCS (50 μg/ml)for 30 min, or untreated, washed and then stimulated with combined PMA(20 ng/ml) and ionophore (1 μM) for 5 min. Cell lysates wereimmunoblotted with anti-ERK-2 mAB

FIG. 10 is a Western blot using anti-phosphotyrosine mAb which showsthat crosslinked anti-CD3ε mAb induces tyrosine phosphorylation ofmultiple proteins. Th0 cells were stimulated with crosslinked anti-CD3εfor 0 to 20 min. Cells were then lysed and postnuclear supernatants weresubjected to SDS-PAGE and Western blotting. Closed symbols denoteanti-CD3εmAb-induced tyrosine phosphorylated proteins.

FIG. 11 is a Western blot using anti-phosphotyrosine mAb whichdemonstrates that CCS inhibits tyrosine phosphorylation inTCR-stimulated T cells. Th0 cells were treated with CCS (0 to 5 μg/ml)for 30 min, washed and then crosslinked anti-CD3ε mAb for 5 min. Cellswere then lysed and postnuclear supernatants were subjected to SDS-PAGEand Western blotting. In this figure, the symbols denote anti-CD3ε mAbinduced tyrosine phosphorylation of ERK-2, which is reduced by CCS.

FIG. 12 is a Western blot using anti-Raf-1 mAb which shows that CCSinhibits the mobility shift of Raf-1. Th0 cells were treated with CCS (0to 50 μg/ml) for 30 min, washed and then stimulated with either (A)combined PMA plus ionophore or (B) then crosslinked anti-CD3ε mAb for 5min. Cells were then lysed and postnuclear supernatants were subjectedto SDS-PAGE and Western blotting.

FIG. 13 is a pair of plots showing that CCS decreases IL-2 productionand proliferation in purified CD4⁺ T cells. T cells were treated withCCS (50 μg/ml), washed and then cultured in either media alone or withimmobilised anti-CD3ε mAb and soluble anti-CD28 mAb. (A) IL-2 productionwas determined by the CTL-L assay as described in Example 5. (B)Proliferation was determined by the incorporation of ³H-thymidine. CD4⁺T cells cultured in the absence of mAb (stimuli) did not produce anydetectable IL-2 and do no proliferate.

FIG. 14 is a pair of plots showing that CCS decreases IL-2 production bysplenocytes but does not inhibit splenocyte proliferation. Splenocyteswere treated with CCS (50 μg/ml), washed and then cultured in eithermedia alone or with immobilised anti-CD3ε mAb. (A) IL-2 production wasdetermined by the CTL-L assay as described in Example 5. (B)Proliferation was determined by the incorporation of ³H-thymidine.Splenocytes cultured in the absence of mAb (stimuli) did not produce anydetectable IL-2 and do no proliferate).

FIG. 15 is a plot which shows that CCS inhibits tumour cell growth invitro. Cancer cell lines were treated with CCS (50, 10, 2.5, 1 and 0.25μg/ml) or water as a control. After 96 h treatment, the effect of CCS ontumour cell growth was evaluated. Columns represent the 50% inhibitoryconcentrations (IC₅₀ μg/ml) of CCS (the amount of CCS required toinhibit 50% of tumour cell growth.

EXAMPLE 1 Purification of Bromelain Proteins

a. Materials

Reagents Bromelain (E.C 3.4.22.4; proteolytic activity, 1,541nmol/min/mg) was obtained from Solvay Inc. (Germany). Fast Flow SSepharose, Pharmalyte 3-10™, Ampholine 9-11™, Ready Mix IEF™(acrylamide, bisacrylamide) and IEF™ markers were obtained fromPharmacia Biotech. Precast 4-20% acrylamide gels and broad rangemolecular weight markers were obtained from Bio-Rad Laboratories. Allother reagents were of analytical grade and obtained from either SigmaChemical Co. or British Drug House.

b. Proteinase Assay

The proteolytic activity of bromelain was determined by use of anin-house microtitre plate based assay using the synthetic substrateZ-Arg-Arg-pNA. This assay was based on that described by Filippova et alin Anal. Biochem., 143, 293-297 (1984). The substrate was Z-Arg-Arg-pNAas described by Napper et al in Biochem. J., 301, 727-735, (1994).

c. Protein Assay

Protein was measured using a kit supplied by Bio-Rad that is a modifiedmethod of Lowry et al (J. Biol. Chem. (1951) 193, 265-275). Samples werecompared to bovine serum albumin standards (0 to 1.5 mg/ml) prepared ineither 0.9% saline or 20 mM acetate buffer pH 5.0, as appropriate.

d. Preparation of Bromelain

All the following steps were performed at ambient temperature (20 to 25°C.). A solution of bromelain (30 mg/ml) was prepared by dissolving 450mg of powder in 15 ml of 20 mM acetate buffer (pH 5.0) containing 0.1 mMEDTA, sodium. The solution was dispensed into 10×1.5 ml microcentrifugetubes and centrifuged at 13,000×g for 10 minutes to remove insolublematerial. The clear supernatants were pooled and used for chromatography

e. Fast Flow S-Sepharose High Performance Chromatography

A Fast flow S-sepharose column was prepared by packing 25 ml of mediainto an XK 16/20™ column (Pharmacia Biotech) and equilibrated with 20 mMacetate buffer (pH 5.0) containing 0.1 mM EDTA on an FPLC™ system at 3ml/min. 5 ml of bromelain solution was injected onto the column. Unboundprotein was collected and the column washed with 100 ml of acetatebuffer. Protein bound to the column was eluted with a linear gradient of0 to 0.8 M NaCl in acetate buffer over 300 ml. 5 ml fractions werecollected throughout the gradient and FIG. 2 shows a typical U.V.chromatogram of crude bromelain obtained from this procedure.

The fractions were then analysed for protein and proteolytic activity asdescribed above and FIG. 3 shows the proteolytic activity against thesynthetic peptide Z-Arg-Arg-pNA and the protein content of theindividual fractions. The protein content profile closely mirrors thatof the U.V., as expected, but the main proteolytic activity is confinedto the two major peaks that correspond to that of bromelain protease(SBP). Small activities are observed in other areas of the chromatogramthat may corresponds to other proteases distinct from SBP, such as thelater eluting CCS fraction, which contains ananain and comosain.

The main peaks identified from the U.V. profile were pooled from threesuccessive runs and named as indicated in Table 1. Pooled fractions wereused for physicochemical characterisation. Pooled fractions wereconcentrated by ultrafiltration and buffer exchanged using PD10 columnsinto isotonic saline (0.9% w/v NaCl). The protein content andZ-Arg-Arg-pNA activity were calculated prior to biological testing andare shown in Table 2.

The pooled fractions were processed for analysis as described below.

TABLE 1 Summary of Pooled Fractions from SP Sepharose HP FractionatedBromelain (QC2322) Fractions Pooled Component Description (Inclusive)CCT Flow through (unbound Unbound column components) flow through CCVFirst peak off column 8–9 CCX Second sharp peak off column 13–14 CCZSmall peak on ascending edge of 19–20 the third main bromelain peak CCYFirst main bromelain peak 23–24 CCW Second main bromelain peak 27–29 CCUSmall peak on descending edge of 33–34 the second main bromelain peakCCS Last double peak off column 39–44

TABLE 2 Calculated Protein Content and Z-Arg-Arg-pNA Activity of PooledFractions used for Testing Biological Activity. Pooled Z-Arg-Arg-pNAActivity Protein Content Fractions (μMoles/min/ml) (mg/ml) CCT 11.301.00 CCV 9.78 1.00 CCX 71.71 1.00 CCZ 688.81 1.00 CCY 1500.0 0.574 CCW1500.0 0.543 CCU 1500.0 0.421 CCS 379.76 1.00f. Processing of Pooled Fractions

The proteolytic activity and protein content of pooled fractions weredetermined and the concentrations adjusted to approximately either 1.4mg/ml of protein or 105 nmoles/min/ml of proteinase activity using aFiltron™ stirred cell containing an ultrafiltration membrane of nominalmolecular weight cut-off of 10 kDa. The fractions were then bufferexchanged using PD10™ columns (Pharmacia Biotech) into isotonic saline(0.9% w/v NaCl), sterile filtered (0.2 μm) and adjusted for proteincontent or proteolytic activity. Samples were then frozen at −80° C. andused in the in vitro studies described below.

g. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Pooled FPLC™ samples were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) on precast 4 to 20% Tgradient gels. Samples were prepared for electrophoresis by acidprecipitation in which 100 μl was mixed with an equal volume of 20% w/vtricloroacetic acid (TCA). Precipitated protein was collected bycentrifugation at 13,000×g for 10 minutes and the supernatant discarded.The pellet was washed twice with 0.5 ml of diethyl ether and left to dryin air at ambient temperature. The pellets were then dissolved in 300 μlof SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8 containing 10% v/vglycerol, 2% w/v sodium dodecyl sulphate and 40 mM dithiothreitol) andheated at 95° C. in a water bath.

SDS-PAGE broad range molecular weight standards diluted 1:20 in SDS-PAGEsample buffer were treated similarly and run with the samples. Gels wererun on a mini Protean II™ electrophoresis system according to Bio-Rad'sprotocol at 240 V and until the dye front reached the end of the gel (30to 45 min).

After electrophoresis, separated proteins were stained overnight withorbital mixing in a solution of 0.075% w/v colloidal brilliant blueG-250 containing 1.5% v/v phosphoric acid, 11.25% w/v ammonium sulphateand 25% v/v methanol. Gels were destained, to obtain a clear background,in a solution of 25% v/v methanol and 10% v/v acetic acid.

Results

The purity of fractions is shown by SDS-PAGE in FIG. 4. All of thepooled fractions except the column flow through (CCT) showed that themajor protein present was of molecular weight between approximately25-28 kDa. This corresponds to the molecular weight of cysteineproteinases isolated from bromelain by other authors (Rowan et al,Methods in Enzymology, (1994), 244, 555-568). The purity of fractionsCCX, CCZ, CCY and CCW appears to be high. Minor components of lowermolecular weight can be observed in some fractions, particularly CCT,CCV, CCX and CCS. Pooled fractions CCU and CCS contain a doublet between25-28 kDa; the higher gel loadings of fractions CCX, CCZ, CCY, and CCWmeans that doublet bands may also be present in these fractions. Asummary of the components and their calculated molecular weights inpooled fractions, as determined by SDS-PAGE, is shown in Table 3.

Proteins in pooled fractions CCX, CCZ, CCY+CCW and CCU were transferredonto nitro-cellulose after SDS-PAGE by Western blotting and probed withrabbit antisera raised against purified stem bromelain protease (SBP)(results not shown). All protein bands in these pooled fractions wererecognised by antibodies in the sera, indicating immunologically similarproteins, probably belonging to the cysteine proteinase family ofenzymes.

TABLE 3 Summary of the Molecular Weights of Proteins found in SPSepharose LIP Pooled Fractions as Determined by SDS-PAGE. PooledMolecular Weight (kDa) of Molecular Weight (kDa) of Fractions MajorProtein Band(s) Minor Protein Band(s) CCT 76.03 15.07 CCV 15.07, 25.85,28.28, 76.03 CCX 25.08 15.07, 76.03 CCZ 27.45 13.37, 16.49, 76.03 CCY27.45 6.5 CCW 27.45 CCU 27.45, 28.28 CCS 15.07, 25.85, 27.45h. Isoelectric Focusing

Pooled fractions (0.5 to 1.0 mg/ml) were diluted 1:3 with deionisedwater and run on gradient gels of pH 3 to 11. Gels were cast using ReadyMix IEF™ to produce a 5.5% T, 3% C polyacrylamide gel containing 10% v/vglycerol, 5.0% Pharmalyte 3-10™ and 2.5% Ampholine 9-11™. Briefly, 10 μlof sample and high PI markers were loaded onto the gel after prefocusingat 700 V. Sample entry was at 500 V for 10 min, focusing was at 2500 Vfor 1.5 hour and band sharpening at 3000 V for 10 min. Afterelectrophoresis the proteins were fixed with a solution of 20% w/v TCAfor 30 min, washed in destain for 30 min to remove TCA and stained withbrilliant blue G-250 as described for SDS-PAGE (see above).

Results

FIG. 5 shows that all fractions except CCX contained basic proteinsfocusing beyond the 9.3 pI marker. Localised charge interactions withthe chromatographic media functional groups may explain why proteins ofpI 3.8 and 3.85 in CCX, adsorbed onto a cation exchange resin at pH 5.0.CCZ was present as a single band of pI 9.7, whilst pooled fractions CCY,CCW, and CCU contained multiple bands of isoelectric points in the rangepH 9.5-9.8. At least part of this heterogeneity can be explained byvariation in the carbohydrate moiety of a common stem bromelain proteinbackbone. The values are in agreement with those reported in theliterature of pI 9.45-9.55 for bromelain (Rowan et al, Methods inEnzymology, (1994), 244, 555-568). Pooled fractions CCS contains twobasic proteins of pI greater than 10.25. Estimates by extrapolation givepIs of 10.4 and 10.45. These correspond to ananain and comosain, and arcin agreement with other estimates (Rowan et al, as above) of pIs greaterthan 10. The pIs of proteins in each of the pooled fractions aresummarised in Table 4.

TABLE 4 Summary of the estimated Isoelectric points of Proteins found inSP Sepharose HP Pooled Fractions. Pooled Fractions Isoelectric Points ofProteins CCT Not detected CCV Not Detected CCX 3.8, 3.85 CCZ 9.7 CCY9.6, 9.7 CCW 9.57, 9.6, 9.7 CCU 9.57, 9.6, 9.75 CCS 10.4, 10.45

EXAMPLE 2 NH₂-terminal Amino Acid Analysis of Bromelain Components

In a separate experiment, pooled fractions of bromelain were run by SDSPAGE and blotted as above onto PVDF membrane. The membrane was stainedwith 0.025% w/v coomassie blue R-250, dissolved in 40% v/v methanol for10 min. followed by destaining in 50% v/v/methanol. The membrane wasdried in air at room temperature and NH₂-terminal amino acid sequencingof the stained proteins was carried out. Briefly, the protein band wascut from the membrane and placed in the upper cartridge of thesequencer. NH₂-terminal amino acid analysis of bromelain components wasdetermined by Edman degradation using a gas phase sequencer (AppliedBiosystems), equipped with an on-line phenylthiohydantion amino acidanalyser. Table 5 shows the first NH₂-terminal amino acids of CCZ (SEQID NO: 1), CCX (SEQ ID NO: 2), stem bromelain protease (SEQ ID NO: 3),ananain (SEQ ID NO: 4) and comosain (SEQ ID NO: 5).

Table 5. NH₂-Terminal Sequence Similarities of CCZ Protein and Those ofKnown Proteinases Isolated form Bromelain.

TABLE 5 NH₂-Terminal Sequence Similarities of CCZ Protein and Those ofKnown Proteinases Isolated from Bromelain. Proteinase Position fromN-Terminus CCZ VLPDSIDWRQKGAVTEVKNRG 1    5    10    15    20 CCXVPQSIDWRDYGAVNEVKN    4    9    14 Stem Bromelain AVPQSIDWRDYGAVTSVKNQNProtease 1     5    10   15   20 Ananain VPQSIDWRDSGAVTSVKNQG1  4    9    14   19 Comosain VPQSIDWRNYGAVTSVKNQG 1  4    9    14   19

All proteins share sequence homologies. Ananain and comosain differ by 2out of 20 amino acids when compared to stem bromelain protease. CCZdiffers by 8 out of 21 amino acids when compared to stem bromelainprotease. CCZ differs from ananain and comosain by 6 out of 20 aminoacids. Comosain differs by 2 amino acids from ananain. Whilst it isclear that these proteins are structurally related, they are alldistinct, showing divergence from each other. These proteinases alsodiffer in their proteinase substrate specificity and their biologicalactivity.

EXAMPLE 3 Fraction CCS Inhibits Tyrosine Phosphorylation of p42 kdaPhosphoprotein.

a. Materials

Antibodies Anti-CD3 ε-chain mAb (145-2C11) and anti-CD28 mAb (PV-1) werepurchased from Pharmingen (San Diego, Calif.) and goat anti-hamster IgGAb was from Sigma (Dorset, UK). Mouse anti-phosphotyrosine mAb (4G10),mouse anti-MAPk R2 (ERK-2) mAb and mouse anti-Raf-1 mAb were from UBI(Lake Placid, N.Y.). Goat anti-mouse and goat anti-rabbit IgG Abconjugated to horse radish peroxidase (HRP) were from BioRad (HemelHemstead, Hertfordshire, UK). Rabbit polyclonal phospho-specific MAPkIgG which recognise tyrosine phosphorylated p44 and p42 MAPks were fromNew England BioLabs (Hitchin, Hertfordshire, UK).

Reagents Phorbol 12-myristate 13-acetate (PMA) and calcium ionophoreA23187 were purchased from Sigma. Bromelain (E.C 3.4.22.4; proteolyticactivity, 1,541 nmol/min/mg) was obtained from Solvay Inc. (Germany).E-64 (L-transepoxysuccinylleucylamido-(4-guanidino)butane, a selectivecysteine protease inhibitor, was from Sigma.

Cells The T cell hybridoma GA15 was a generous gift from B. Fox(ImmuLogic Pharmaceutical Corporation, Boston, Mass.). GA15 wasgenerated by fusing the thymoma BW5147 with the T_(h)2 clone F4 specificfor KLH in association with I-A^(b), and were maintained as previouslydescribed (Fox, 1993, Int. Immunol., 5, 323-330). GA15 exhibit a T_(h)0cell phenotype as they produce IL-2, IL-4 and IFN-γ followingstimulation with crosslinked anti-CD3ε mAb (Fox, 1993).

b. Stimulation of T cells. Cells (2×10⁷) suspended in RPMI 1640 weretreated with CCS (1 to 50 μg/ml) diluted in saline (0.9% (w/v)) for 30min at 37° C. Mock treated cells were treated with an equal volume ofsaline (diluent). At high concentrations of CCS (50 or 100 μg/ml) cellaggregation occurred, as noted previously in studies with crudebromelain. Following treatment, cell aggregates were gently dispersed bywashing cells 3 times and then resuspending in fresh RPMI. Cells werestimulated via the cell surface with crosslinked mAb to the TCR(anti-CD3ε), or directly, using combined PMA (20 ng/ml) and ionophore (1μM) for times indicated in figure legends and the text.

Stimulation via the TCR was conducted by first incubating T cells on icefor 30 min with anti-CD3ε mAb (20 μg/ml). Excess mAb was then removed bywashing once at 4° C. and anti-CD3ε mAb was crosslinked with goatanti-hamster IgG (20 μg/ml) at 37° C. Stimulation was terminated by theaddition of ice-cold lysis buffer (25 mM Tris, pH 7.4, 75 mM NaCl, 2 mMEDTA, 0.5% Triton X-100, 2 mM sodium orthovanadate, 10 mM sodiumfluoride, 10 mM sodium pyrophosphate, 74 μg/ml leupeptin, 740 μM PMSFand 74 μg/ml aprotinin) for 30 min with continual rotation at 4° C.Lysates were clarified (14,000×g for 10 min) and an equal volume of2×SDS-PAGE sample buffer (50 mM Tris, pH 7, 700 mM 2-ME, 50% (v/v)glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) was added topostnuclear supernatants. Proteins were solubilised at 100° C. for 5 minand samples containing 1×10⁶ cell equivalents were resolved by SDS-PAGE.

c. Immunoblotting. Separated proteins were transferred to nitrocellulosemembranes (Bio-Rad) which were then blocked with 5% (w/v) bovine serumalbumin (Sigma, fraction V; BSA), 0.1% Nonidet p-40™ in Tris-bufferedsaline (170 mM NaCl and 50 mM Tris, pH 7.4; TBS). Immunoblots wereincubated with the appropriate antibodies as indicated in figurelegends. Primary antibodies were diluted in antibody dilution buffercomprised of 0.5% (w/v) BSA, 0.1% (v/v) Tween-20 in TBS at 4° C. for 2 hfollowed by detection with the appropriate secondary antibody conjugatedto horseradish peroxidase diluted in antibody dilution buffer at 4° C.for 1 h. Following each incubation step, membranes were washedextensively with 0.1% Tween-20 in TBS. Immunoreactivity was determinedusing the ECL chemiluminescence detection system (Amersham Corp.,Arlington Heights, Ill.).

d. Inhibition of proteolytic activity of CCS. A specific cysteineprotease inhibitor, E-64, was used to inactivate the proteolyticactivity of CCS. CCS (25 μg/ml) diluted in 3 μM dithiothreitol, 100 μME-64, 60 mM sodium acetate (pH 5) was incubated for 10 minutes at 30° C.The inactivated CCS was then dialysed overnight in saline at 4° C.Earlier studies with crude bromelain have shown that these conditionsare sufficient to induce 99.5% inactivation of proteolytic activity asassayed with the Z-Arg-Arg-pNA substrate (see above). T cells weretreated with E-64 inactivated CCS (25 μg/ml) and compared with untreatedCCS and mock-treated T cells stimulated with PMA plus ionophore.

Results

a. Fraction CCS inhibits tyrosine phosphorylation of p42 kdaphosphoprotein. We have previously shown that bromelain blocks tyrosinephosphorylation of ERK-2 following stimulation of T cells with combinedPMA plus calcium ionophore (WO-A-96/00082). Phorbol ester and ionophorestimulation of T cells act synergistically to reproduce many features ofTCR stimulation such as IL-2 secretion, IL-2 receptor expression, and Tcell proliferation (Truneh et al., 1985, Nature, 313, 318-320; Rayter etal., 1992, EMBO, 11, 4549-4556). Phorbol esters can mimic antigenreceptor triggering and bypass TCR-induced protein tyrosine kinases toactivate ERK-2 by a direct agonist action on PKC and p21^(RaS). Calciumionophore A23187 induces increased intracellular release of Ca²⁺ andtherefore mimics the action of inositol 1,4,5-trisphosphate (IP₃).Phorbol esters and ionophore however, stimulate PKC pathways that arenot controlled by the TCR (Izquierdo et al., 1992, Mol. Cell. Biol., 12,3305-3312) suggesting separate intracellular pathways within T cellsthat regulate T cell function. We therefore investigated which fractionof bromelain could block T cell signalling via the TCR-independentpathway by examining its effect on PMA and ionophore-induced tyrosinephosphorylation.

Stimulation of T cells with combined ionophore and PMA induced tyrosinephosphorylation of several proteins including those of circa 100 kda, 85kda, 42 kda and 38 kda. CCS (50 μg/ml) pre-treatment reduced tyrosinephosphorylation of the p42 kda protein, and did not significantlyinhibit phosphorylation of any other substrate (FIG. 6). In twoexperiments, CCS, but no other fraction, also increased tyrosinephosphorylation of circa 36 kda, 38 kda, 85 kda, 94 kda and 102 kdaproteins (FIG. 7 and FIG. 8).

The ability of CCS to block tyrosine phosphorylation of the 42 kdaphosphoprotein was dose-dependent (FIG. 8) and dependent on itsproteolytic activity, since E-64 completely abrogated the inhibitoryeffect of CCS on p42 kda phosphorylation (FIG. 8). E-64 treatment of Tcells did not affect PMA and ionophore-induced T cell signalling.

CCS inhibits ERK-2 tyrosine phosphorylation. We suspected that the 42kda phosphoprotein inhibited by CCS was the MAPk ERK-2, so we conductedimmunoblot analysis with specific anti-ERK-2 mAb and anti-phospho MAPkantibodies, which specifically detects ERK-1 and ERK-2 only whencatalytically activated by phosphorylation at Tyr204. Immunoblotting ofCCS-treated cells that had been stimulated with PMA plus ionophore,confirmed that the p42 kda phosphoprotein was indeed ERK-2 (FIG. 9).

CCS reduces TCR-induced tyrosine phosphorylation of ERK. We nextinvestigated the effect of CCS on TCR-mediated signal transduction byassessing substrate tyrosine phosphorylation of GA15 stimulated withcrosslinked anti-CD3ε mAb. Immunoblots of GA15 lysates, using specificanti-phosphotyrosine mAb, revealed increased tyrosine phosphorylation ofmultiple proteins including those of circa 120 kda, 100 kda, 85 kda, 76kda, 70 kda, 42 kda and 40 kda, consistent with phosphoproteins observedin other T cell lines following TCR-ligation (June et al, 1990, J.Immunol., 144, 1591-1599 and Proc. Natl. Acad. Sci. USA, 87, 7722-7726,reviewed by Cantrell, 1996, Annu. Rev. Immunol., 14, 259-274) (FIG. 10).Tyrosine phosphorylated proteins were readily detected between 2 and 5min following stimulation and remained phosphorylated for at least 10min (FIG. 10). GA15 cells stimulated with anti-CD3 mAb alone orcross-linking Ab, did not induce tyrosine phosphorylation of anycellular substrate (data not shown). Again, CCS pretreatment of GA15 for30 min caused a reduction in TCR-induced protein tyrosinephosphorylation of ERK-2 in a dose-dependent manner (FIG. 11). CCS didnot markedly affect tyrosine phosphorylation of other TCR-inducedphosphoproteins, suggesting that CCS has a selective mode of action.

EXAMPLE 4 CCS Retards the Mobility Shift of Raf-1

Raf-1 is an immediate upstream activator of MEK-1 which activates ERK-2.Raf-1 activation requires phosphorylation on specific serine andthreonine residues (Avruch et al., 1994, TIBS, 19, 279-283). Toinvestigate whether CCS affects any other substrates upstream from ERK-2in the MAP kinase cascade, we investigated the effect of CCS on Raf-1. Tcells were treated with CCS (0 to 50 μg/ml) and then stimulated witheither anti-CD3ε, mAb or combined PMA plus ionophore as describedearlier. Results show that CCS blocks the mobility shift of Raf-1,indicating that it blocks its protein phosphorylation and thusactivation. This data confirms that CCS has an effect on the MAP kinasecascade (FIG. 12) and that the effect of CCS may not be directly onERK-2, but on upstream substrates in the MAPk cascade.

EXAMPLE 5 Effect of CCS on IL-2 Production and T cell Proliferation

a. Materials

Cells Splenocytes were isolated from female BALB/c mice (6-8 weeks old),as previously described in WO-A-96/00082. Highly purified CD4⁺ T cellswere isolated from splenocytes using magnetic activated cell sorting(MACS).

b. Interleukin 2 production. T cells diluted in RPMI were treated withCCS (50 μg/ml) or saline at 37° C. for 30 min, washed in fresh RPMI andthen resuspended in culture medium. T cells were stimulated to producecytokine mRNA by immobilised anti-CD3ε (4 μg/ml) and soluble anti-CD28(10 μg/ml). Anti-CD3ε mAb diluted in PBS was immobilised to 24-well,flat bottom, microculture plates (Corning, Corning, N.Y.) by incubationfor 16 hours at 4° C. Wells were then washed three times in PBS prior toaddition of triplicate cultures of either splenocytes or purified CD4⁺ Tcells (2.5-5×10⁶ cells per well) which were incubated at 37° C. inhumidified 5% CO₂ for 24 h. IL-2 levels in the culture supernatant weremeasured using the CTL-L bioassay (Gillis et al., 1978, J. Immunol.,120, 2027-2032).

c. T Cell Proliferation. T cells were treated with CCS (50 μg/ml) for 30min, washed in RPMI then stimulated with immobilised anti-CD3ε mAb aloneor combined anti-CD3ε mAb plus anti-CD28 mAb. Cells were then culturedin 96 well, flat-bottom plates (Nunc) at 10⁵ cells per well for 36 h.Cultures were pulsed with 0.5 μCi of [³H]TdR 12 h prior to harvestingonto glass fibre filters.

Results

a. CCS inhibits IL-2 production and proliferation of CD4⁺ T cells.Activation of p21^(Ras), Raf-1, MEK-1 and ERKs are essential forinduction of IL-2 transcription in T cells (Izquierdo et al., 1993, J.Exp. Med., 178, 1199). IL-2 is the major autocrine T cell growth factorwhich induces proliferation of T cells. The defect in ERK activationdemonstrated here could therefore be expected to inhibit IL-2 productionand T cell proliferation. We therefore investigated whether CCS couldeffect a functional outcome of T cell signalling, namely IL-2 productionand proliferation in murine splenocytes and highly purified CD4⁺T cells.CCS (50 μg/ml) treatment of purified CD4⁺T cells reduced both IL-2production and proliferation when the ERK pathway was stimulated withanti-CD3ε mAb (FIGS. 13 a and 13 b). CCS also blocked IL-2 production bysplenocytes, however it did not affect splenocyte proliferation (FIGS.14 a and 14 b), suggesting that an as yet unidentified component in CCSwas acting on accessory cell populations in splenocyte cultures, such asB cells or macrophages. Bromelain can increase costimulatory signals toT cells via an action on B cells. Regardless of the putative effect ofCCS on accessory cells, data clearly indicate that CCS blocks IL-2production and proliferation of purified CD4⁺ T cells, suggesting thatCCS blocks T cell activation. IL-2 production and proliferation weredependent on cell stimulation with anti-TCR antibodies as no cytokinewas detected in cells cultured in tissue culture media alone (FIGS. 13and 14).

EXAMPLE 6 Effect of CCS on Human Tumour Cell Growth In Vitro

a. Materials

Cells Tumour cell lines were provided by L. Kelland (Institute of CancerResearch, Sutton, UK) and were as follows; ovarian (SKOV-3, CH-1,A2780), colon (HT29, BE, LOVO), breast (MCF-7, MDA231, MDA361), lung(A549, CORL23, MOR) and melanoma (G361, B008, SKMe124).

b. Growth Inhibition of Human Tumour Cell Lines. Studies were conductedby L. Kelland (Institute of Cancer Research, Sutton, UK). Cell lineswere trypsinised and single viable cells were seeded into 96-wellmicrotitre plates at a density of 4×10³ cells/well in 160 μl growthmedium. After allowing for attachment overnight, CCS was then added toquadruplicate wells in 40 μl of growth medium to give a range of finalconcentrations in wells of 50, 10, 2.5, 1 and 0.25 μg/ml. Eight wellsserved as control, untreated wells. CCS was diluted immediately prior toaddition to cells in sterile water. CCS exposure to cells was for 96 hwhereupon the cell number in each well was determined by staining with0.4% sulforhodamine B in 1% acetic acid as described previously (Kellandet al., 1993, Cancer Res., 53, 2581-2586). 50% inhibitory concentrations(IC₅₀ values in μg/ml) were then calculated from plots of concentrationversus control (%) absorbance (read at 540_(nm).).

Results

a. CCS inhibits human tumour growth in vitro. p21^(Ras) and Raf-1 areimportant oncogenes, which when mutated cause uncontrolled cell growthand proliferation, leading to cancer. Since we have shown that CCS canblock the effects of the p21^(Ras)/Raf-1/MEK1/ERK kinase signallingcascade, we investigated whether CCS could block tumour growth. CCStreatment of human tumour cells resulted in a reduction in the growth ofseveral different ovarian, lung, colon, breast and melanoma tumour celllines in vitro (FIG. 15). CCS did not affect all cell lines equally,suggesting that CCS has a selective action.

1. A method for the treatment of an inflammatory disease or aninflammatory condition in a patient in need of such treatment, saidmethod comprising either enteral or parenteral administration to saidpatient of an effective amount of an agent, wherein said agent is acomponent of bromelain comprising proteins having molecular weights ofabout 15.07 kDa, 25.85 kDa, and 27.45 kDa as determined by SDS-PAGE andhaving isoelectric points of 10.4 and 10.45, wherein said component ofbromelain does not comprise a protein comprising an N-terminal aminoacid sequence of SEQ ID NO: 1 and having an isoelectric point of 9.7,and wherein said component of bromelain is obtainable by a methodcomprising: (i) dissolving bromelain in acetate buffer at pH 5.0, (ii)separating the components of the bromelain by fast flow high performanceliquid chromatography on S-sepharose eluted with a linear gradient of 0to 0.8 M sodium chloride in acetate buffer over 300 ml, (iii) collectingthe fraction corresponding to a final double peak off the column, and(iv) isolating proteins from the fraction collected in (iii), whereby aprotein comprising an N-terminal amino acid sequence of SEQ ID NO: 1 andhaving an isoelectric point of 9.7 is absent from said component ofbromelain, to thereby inhibit or suppress T cell activation in saidpatient, such that said inflammatory disease or inflammatory conditionin said patient is treated.
 2. The method of claim 1, wherein inhibitionor suppression of T cell activation is through inhibition of amitogen-activated protein kinase pathway.